APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G) has been identified as a key component in restricting the zoonotic transmission of retroviruses to humans. Excitingly, it appears that the antiviral activity of APOBEC3G could be extended to restrict the infection of viruses, such as HIV, that the human immune system and modern medicine have yet to conquer. Of the six accessory proteins encoded by the HIV genome, one protein, Vif (viral infectivity factor), performs the specific function of inhibiting APOBEC3G antiviral activity. In the absence of Vif, APOBEC3G is incorporated into HIV virions, and inhibits further HIV replication through hypermutation of the viral genome and/or a second activity linked to APOBEC3G binding to viral RNA. Vif is primarily believed to inhibit APOBEC3G by targeting it for degradation via the ubiquitin/proteosome pathway, but recent evidence suggests that inhibition can occur without degradation. The purpose of this proposal is to obtain a structural understanding of how Vif is able to inhibit APOBEC3G function. The interaction between these two proteins has been know for many years but in vitro studies of both proteins have been hampered by the inability to acquire large amounts of soluble, active protein. 1) Work from this proposal will first seek to generate a system allowing for expression and purification of mg amounts of soluble, active APOBEC3G. To do this, an E. coli screening system will be employed in which APOBEC3G mutants with enhanced solubility will be detected through the fluorescence of a GFP reporter fusion. 2) The second goal is to obtain the structure of APOBEC3G. Crystal trials will be conduted as a more rapid method to obtain structural data, but the size of APOBEC3G, 46 kD, makes an NMR structure also possible. 3) To detevelope a molecular understanding of the interaction between Vif and APOBEC3G, the two proteins will be cocrystalized as the size of the complex, 70 kD, is too large for NMR studies. Historically, Vif and APOBEC3G have been prone to nonspecifically interact with other proteins and precipitate from solution. Coexpression of a more soluble form of APOBEC3G with Vif will encourage formation of specific interactions leading to soluble complexes and and minimize the nonspecific interactions that lead to precipitation. Also, the ability of APOBEC3GA/if complexes to bind important factors for the incorporation of APOBEC3G into virions, such as RNA and viral nucleocapsid, will be examined. Public Health Relevance: The structural data obtained from this proposal will serve as a model for the screening and/or design of new drugs to inhibit the interaction of Vif with APOBEC3G and therefore inhibit HIV spread among individuals as well as progression of HIV infected individuals to AIDS.